Engineered Microbes Make Plant Hormones a Sustainable Harvest

As seeds grow, their roots first spread out in the soil before green shoots push through the earth, guided by plant hormones that regulate development and environmental responses. One class of hormones, strigolactones (SLs), act as signaling molecules to influence growth and development.¹ Roots also exude SLs in a symbiotic relationship with soil microbes to address nutrient deficiencies. While the functions of this array of SLs are well understood, the evolution of their biosynthesis remains less explored.

Studying SLs is challenging because they are found in trace levels in plants, requiring up to 1,000 liters of xylem sap. To investigate a different approach, Yanran Li, a synthetic biologist at the University of California, San Diego, incorporated optimized alternative pathways into Escherichia coli and Saccharomyces cerevisiae strains to mass-produce SLs. In a study recently published in Science, Li and her colleagues at the University of California, Riverside, and Utsunomiya University reported that these microbial factories enabled easier characterization of proteins involved in SL production. These findings could help researchers better understand SLs’ role in plant adaptation and survival.²This microbial platform not only makes SL production scalable but also opens new opportunities to explore how different genes and enzymes contribute to the biosynthesis of these versatile molecules.

Plant SLs serve two roles: canonical ones act as external signaling molecules typically released from the roots, while noncanonical SLs function as hormones within the plant. About 30 SLs have been discovered so far, and they share a common precursor, carlactonic acid (CLA). The cytochrome P450 enzyme CYP722C drives the conversion of CLA to many different SLs in flowering plants.

However, Li and her team sought to explore the evolution of SL biosynthesis by investigating related proteins to CYP722C. They hypothesized that uncharacterized sister enzymes, such as CYP722A and CYP722B, found across seed plants, might also contribute to SL production and play essential roles in plant physiology.

To test this, they engineered E. coli and S. cerevisiae strains to express known SL biosynthesis pathway genes, as well as the CYP722A/B genes, from 16 different seed plant species, including poplar, pepper, pea, and peach. Then, the researchers grew these microbes in flasks, transforming them into tiny SL factories. To ensure sufficient production for study, the team optimized their microbial mixtures by testing different gene variants and fermentation conditions, resulting in yields more than 125 times higher than those achieved with their previous microbial methods.

“By using this microbial cell factory, you can bypass extracting tons of xylem sap and thus destroying dozens of trees to discover the molecules important for the physiology of plants,” said Li in a press release.

Image of Kaibiao Wang, a postdoctoral researcher at UC San Diego and one of the study coauthors. He wears safety glasses and a blue lab coat. He is holding a pipette above a flask filled with yellow liquid. — The researchers harvested large amounts of strigolactones from flasks of co-cultured *E. coli* and yeast. These microbial cell factories enabled them to bypass the laborious method of collecting tree sap.
David Baillot, UC San Diego Jacobs School of Engineering

To the researchers’ surprise, they found that when the microbes expressed CYP722A and CYP722B, they produced a novel compound 16-hydroxy-carlactonoic acid (16-OH-CLA), a less-studied noncanonical SL derived from CLA. While 16-OH-CLA was previously identified in the model plant Arabidopsis thaliana, its synthesis and specific function remained a mystery.³ This piqued Li’s interest.

To investigate further, the team tested whether CYP722A was essential for producing 16-OH-CLA in living plants. They measured 16-OH-CLA levels in wild type, SL-deficient, and CYP722A knockout Arabidopsis thaliana. While most known SLs are typically found in roots, 16-OH-CLA was primarily detected in shoots of wild type plants. This pattern held true across other plants, including poplar, pea, and pepper. During early development, 16-OH-CLA accumulated in branching shoots but was absent in roots, with its levels decreasing as the plants matured. However, 16-OH-CLA levels were undetectable in SL-deficient and CYP722A plants, supporting the role of CYP722A driving the production of 16-OH-CLA.

But how did this pathway relate to CYP722C? Phylogenetic and structural analyses revealed that CYP722A is the evolutionary precursor to CYP722C, which diverged by a single mutation. This mutation altered CLA’s oxidation site, directing SL production to roots (canonical pathway) or shoots (noncanonical pathway).

This study underscores key aspects of plant hormone biology and offers practical tools for investigating trace compounds like SLs, making them easier to study. By leveraging microbial cell factories, scientists can dig deeper into how these molecules shape plant growth and resilience, paving the way for agricultural innovations.